Multichannel analysis and monitoring of multiple gene expressions by using bioluminescent reporters

通过检测编码荧光素酶的报告基因，我们监测了单细胞中多个基因的表达。该方法被应用于二维和时间过程分析，以可视化活细胞内分子的相互作用。(1)新型生物发光报告基因的克隆及多色报告载体的构建铁路蠕虫的两种发光酶催化同一底物分别发出红光和绿色光。通过插入cDNA构建双报告载体，一个在加压素下，另一个在CMV启动子下。虽然可以检测到光发射，但哺乳动物细胞中的发光很弱。因此，具有更高强度的双报告基因构建使用瓦古拉希氏虫荧光素酶和萤火虫荧光素酶。将生长激素（GH）基因启动子序列连接到vargula荧光素酶cDNA的上游，并将报告载体转染到GH 3细胞中。的st ...更多信息 将萤火虫荧光素酶进一步转染GH3细胞的可克隆。分别从GH3细胞连续监测来自vargula和萤火虫荧光酶的发光。(2)多色监测系统的构建：我们构建了一个由37 ℃的暗箱组成的检测系统，其中一个装有6个草底培养皿的转台在冷却的CCD摄像机上旋转。在通过液晶过滤器分离出具有峰值波长的光子后，发光二极管对光子进行计数。(3)启动子序列的克隆和活细胞中靶基因表达的连续监测：使用与GFP或萤火虫荧光素酶cDNA连接的小鼠Per1和BMAL1基因的不同长度的启动子序列分析转录调控。我们鉴定了该基因启动子的调控区，并构建了双色报告基因来监测Per1和BMAL1的表达。我们正在构建转基因小鼠系，以研究分子时钟机制，监测这两个时钟基因的表达。少

By transfecting reporter genes which encode luciferase emitting light with different wave lengths, we monitored expression of multiple genes in single cells. The method was applied for two dimensional and time-course analyzes to visualize the interactions of molecules within living cells.(1) Cloning of novel bioluminescence reporter genes and construction of multi-color reporter vector : Two luciferases of the railroad-worm emit red and green lights by catalyzing the same substrate. The dual reporter vector was constructed by inserting the cDNAs, one under the vasopressin and the other under CMV promoter. Although the light emission could be detected, the luminescence in the mammalian cell was weak. Therefore, dual reporter with higher intensity was constructed using vargula hilgendorfii luciferase and firefly luciferase. Promoter sequence of growth hormone (GH) gene was connected to the upstream of vargula luciferase cDNA and the reporter vector was transfected to the GH3 cell. The st … More able colony of GH3 cells was further transfected firefly luciferase. Luminescence from vargula and firefly luciferases was separately monitored from GH3 cells continuously.(2) Construction of multi-color monitoring system : We constructed the detection system consisting of dark box kept 37 ℃ in which a turn table carrying 6 grass-bottom petri dishes rotates on the cooled CCDcamera. A luminocapter counted the photons after separating those with the peak wavelength through liquid crystal filters.(3) Cloning of promoter sequences and continuous monitoring of targeted gene expression in living cells : Transcriptional regulation was analyzed using various length of promoter sequences of mouse Per1 and BMAL1 genes connected with either GFP or firefly luciferase cDNA. We identify the regulatory region in the promoter and constructed dual color reporter to monitor Per1 and BMAL1 expressions. We are constructing the transgenic mice line to study the molecular clock mechanisms monitoring the expression of the two clock genes. Less